Histamine receptor H3 polynucleotides

ABSTRACT

Novel splice variants of the human H3 histamine receptor are described. The splice variants have deletions of portions of the N-terminal of the wild type H3 receptor. The splice variants are useful in methods for identifying agonists, inverse agonists or antagonists of histamine action at the H3 receptor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application under 35 U.S.C. §371 of PCT Application No. PCT/US02/35375, filed Nov. 4, 2002, whichclaims priority under 35 U.S.C. § 119 from U.S. Application No.60/333,094 filed Nov. 13, 2001.

FIELD OF THE INVENTION

The present invention relates to human histamine receptors, particularlyto variant forms of the H3 receptor.

BACKGROUND OF THE INVENTION

Histamines are implicated in a number of medical conditions, includinginflammation, asthma, allergy, atopic dermatitis, stroke, myocardialinfection, migraine, chronic obstructive pulmonary disease (COPD),rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease,and psoriasis. Histamines regulate the intensity and duration of immuneresponses and are involved in cell-to-cell communication. Histamines arealso involved in leukocyte migration and bronchovasoconstriction. Asestablished by radioligand binding, physiological assays, and molecularcloning, different types of receptors for histamines exist. Furthermore,specific histamine receptor subtypes are involved in specific medicalconditions such that drugs with subtype selectivity can be utilized totarget individual medical conditions.

At present there are four known human histamine receptors, H1, H2, H3and H4, all of which are G-protein coupled molecules. Although theexistence of histamine receptors had been established pharmacologicallyfor decades, the H1 and H2 receptors were cloned only in 1991 (Yamashitaet al. 1991 Proc. Natl. Acad. Sci. USA 88:11515; Gantz et al. 1991 Proc.Natl. Acad. Sci. USA 88:429), and the H3 receptor and the H4 receptorwere not cloned until more recently (Lovenberg et al. 1999 J. Mol.Pharmacol. 55:1101; U.S. Pat. No. 6,136,559; WO 00/20011(PCT/US98/21090); Oda et al. 2000 J. Biol. Chem. 275:36781; Zhu et al.2001 Mol. Pharmacol. 59:434; Liu et al. 2001 Mol. Pharmacol. 59:420;Morse et al. 2001 J. Pharm. Exp. Ther. 296:1058; as well as U.S. Pat.No. 6,204,017 and WO 01/25432 A2 (PCT/US00/27481); WO 01/46414 A1(PCT/JP00/09038); and Nguyen et al. 2001 Mol. Pharmacol. 59:427).

The H3 receptor subtype has been found in both the central nervoussystem and in the peripheral nervous system as a presynaptic receptorcontrolling the release of histamine and of several neurotransmitters.The CNS effect of the H3 antagonists makes them potential candidates forthe treatment of obesity, epilepsy, depression, sleep/wake disorders andage-related memory disorders, such as Alzheimer's disease andattention-deficit hyperactivity disorders. The negative feedbackmechanism on sensory C fibres and the resultant anti-inflammatoryeffects of H3 receptor agonists suggests a possible role for thesecompounds in treatment of migraine, asthma, cardiac disorders andneurogenic airway inflammation. Various antagonists of the H3 histaminereceptor and methods for their use for treating CNS disorders, includingAlzheimer's disease, narcolepsy, and comas induced by stroke, drugs oralcohol, for suppressing appetite in the treatment of obesity, and fortreatment of allergy, inflammation, cardio and cerebro vasculardiseases, gastrointestinal disorders, psychiatric disorders, sleepdisorders and hypothalnic dysfunction are disclosed in U.S. Pat. Nos.5,380,858; 5,486,526; 5,633,382; 5,639,775; 5,652,258; 5,990,317;6,008,240; 6,072,057; and 6,166,060; see also U.S. Pat. No. 6,136,559;WO 00/20011 (PCT/US98/21090).

Recent molecular studies have shown that a single form of the H3 genecan give rise to multiple mRNA isoforms in rat (Drutel et al. 2001 Mol.Pharmacol. 59:1) and in guinea pig (Tardival-Lacombe et al. 2000Neuroreport 11:755). In humans, six splice variants of the H3 receptorwere recently reported in the thalamus (Coge et al. Biochem. J. 2001355:279). These variants were found to be coexpressed in human brain,but their relative distribution varied in a region-specific manner. Thevariants displayed deletions in either the putative second transmembranedomain or in the third intracellular loop.

In view of the important role that histamines play in many physiologicalprocesses and medical conditions, there is a need for materials andmethods, including novel histamine receptors, useful for theidentification of agonists, inverse agonists and antagonists selectivefor specific types of histamine receptors.

SUMMARY OF THE INVENTION

The present invention provides novel variants of the H3 histaminereceptor. In one aspect, the present invention provides polypeptidescomprising the novel receptor variants, including fragments and analogsthereof. In another aspect are provided polynucleotides encoding thenovel H3 receptor variants, including fragments and analogs thereof, aswell as antisense nucleic acids, recombinant vectors comprising thepolynucleotides and host cells comprising the vectors. Methods of makingpolypeptides according to the invention by culturing the host cell underconditions for expressing such polypeptides are also provided. In afurther aspect, the invention provides antibodies, including monoclonalantibodies, that are specific for the novel polypeptides according tothe invention, including variant isoforms of the H3 receptor. Methods ofidentifying inhibitors of histamine binding to the polypeptidescomprising the H3 receptor variants, including fragments and analogsthereof, as well as of identifying modulators such as agonists,antagonists or inverse agonists of such polypeptides are all providedherein. Such modulators identified in the assays disclosed herein areuseful, for example, as therapeutic agents, and diagnostic agents.Indications for said therapeutic agents include, but are not limited to,central nervous system disorders, for example depression, anxiety,psychoses (for example schizophrenia), tardive dyskinesia, Parkinson'sdisease, obesity, hypertension, Tourette's syndrome, sexual dysfunction,gastrointestinal disorders, psychiatric disorders, sleep/wake disorders,hypothalamic dysfunction, drug addiction, drug abuse, cognitivedisorders, Alzheimer's disease, senile dementia, narcolepsy, comasinduced by stroke, drugs or alcohol, obsessive-compulsive behavior,panic attacks, pain, social phobias, eating disorders and anorexia,appetite suppression for treatment of obesity, cardiovascular andcerebrovascular disorders, non-insulin dependent diabetes mellitus,hyperglycemia, constipation, arrhythmia, disorders of theneuroendrocrine system, stress, and spasticity, as well as acidsecretin, ulcers, airway constriction, asthma, allergy, inflammation,and prostate dysfunction.

Additional aspects of the invention will be apparent from thedescription herein.

DETAILED DESCRIPTION

The present invention provides novel splice variants of the human H3histamine receptor. Novel splice variants of the human H3 receptor havebeen isolated and shown to contain a deletion of a region of theN-terminal extracellular portion of the H3 wild type receptor fromresidues 7 through 42. The H3g variant (SEQ ID NO: 13 and 14) and theH3h variant (SEQ ID NO: 17 and 18) were identified by PCR amplificationof cDNA prepared from human brain mRNA. The H3g variant contains a 108base deletion in the region coding for the N-terminal compared to thesequence of wild type H3 receptor (wild type H3 receptor is designatedherein as H3a, amino acid sequence SEQ ID NO:2, polynucleotide sequenceSEQ ID NO:1). The deletion in the H3g variant cDNA results the loss of36 amino acid residues (residues 7–42 of the wild type sequence) in theextracellular N-terminal portion prior to transmembrane region 1 (TM1).The H3h variant contains the same 108 base deletion in the N-terminalcoding region and an additional 240 base deletion in the region codingfor the i3 intracellular loop. A splice variant containing a similar 240base deletion was recently described by Coge et al. (Biochem. J. 2001355:279) as H_(3(Δi3,365aa)). Other novel H3 receptor variantscontaining the 108 base deletion in the N-terminal coding region plusadditional deletions are provided as H3i (SEQ ID NO:15 and 16), H3j (SEQID NO:19 and 20), H3k (SEQ ID NO:21 and 22), and H3l (SEQ ID NO:23 and24). Each of these contains the 108 base N-terminal deletion describedherein and a previously described deletion (Coge et al. 2001, supra) inthe region encoding the carboxy terminal portion of the receptor. Inaddition to the 108 base, 36 residue N-terminal deletion, H3i contains a42 base, 14 residue deletion in transmembrane region 2 (TM2), H3jcontains a 90 base, 30 residue deletion in intracellular loop 3 (i3),H3k contains a 348 base, 116 residue deletion in intracellular loop 3(i3), and H3l contains a 357 base, 119 residue deletion in intracellularloop 3 (i3). The sequences of the previously described carboxy terminaldeletion variants are provided as SEQ ID NOs: 4, 6, 8, 10 and 12, withcorresponding nucleic acid sequences SEQ ID NOs: 3, 5, 7, 9, and 11.

This invention thus provides novel histamine H3 receptor variants,isolated polynucleotides encoding the receptor variants, and recombinantvectors and host cells comprising such polynucleotides. Antisensenucleic acids for the H3 receptor variants are also provided. Thehistamine receptor variants can be actively expressed in mammalian cellswhere they will display active ligand binding and positive or negativeintracellular signaling upon ligand activation. These novel receptorvariants have measurable affinity for histamine. This invention furtherprovides methods for the discovery of selective agonists, antagonists orinverse agonists of the H3 receptor or receptor variants that may beuseful in the treatment and management of a variety of diseasesincluding, for example, central nervous system disorders, for exampledepression, anxiety, psychoses (for example schizophrenia), tardivedyskinesia, Parkinson's disease, obesity, hypertension, Tourette'ssyndrome, sexual dysfunction, gastrointestinal disorders, psychiatricdisorders, sleep disorders, hypothalamic dysfunction, drug addiction,drug abuse, cognitive disorders, Alzheimer's disease, senile dementia,narcolepsy, comas induced by stroke, drugs or alcohol,obsessive-compulsive behavior, panic attacks, pain, social phobias,eating disorders and anorexia, appetite suppression for treatment ofobesity, cardiovascular and cerebrovascular disorders, non-insulindependent diabetes mellitus, hyperglycemia, constipation, arrhythmia,disorders of the neuroendrocrine system, stress, and spasticity, as wellas acid secretin, ulcers, airway constriction, asthma, allergy,inflammation, and prostate dysfunction. Further provided are methods fortreating histamine-mediated medical conditions comprising administeringto a mammal afflicted with a medical condition caused or mediated byhistamine, an effective amount of an agonist, inverse agonist orantagonist of the histamine receptor that binds to an H3g, H3h, H3i,H3j, H3k, or H3l receptor, for example, having an amino acid sequencedefined by SEQ ID NO:14, 18, 16, 20, 22 or 24, or a subsequence thereof,and pharmaceutical compositions comprising one or more of such agonist,inverse agonist or antagonist and a pharmaceutically acceptable carrier.Preferably, the mammal is a human being.

“Polypeptide” refers to a polymer of amino acids joined by conventionalpeptide bonds and includes proteins and peptides of any size, bothglycosylated and non-glycosylated, pegylated and non-pegylated, and allother modified forms that retain the same primary amino acid sequence asthe unmodified form.

“Ligand” refers to a molecule capable of binding to H3 receptorsaccording to the invention. Thus histamine itself is a ligand, as areagonists, inverse agonists and antagonists that may compete withhistamine for specific binding to the receptors.

“Histamine surrogate” refers to a compound that acts as a ligand for ahistamine receptor and modulates the activity of the histamine receptorin a similar fashion to the natural ligand histamine. Examples ofhistamine surrogates include amitriptyline, chlorpromazine, doxepin,cinnarizine, promethazine, cyproheptadine, clemizole, mianserin,clozapine, chlorpheniramine, imetit, pheniramine, dimaprit, α-methylhistamine or cimetidine.

“Agonist” refers to a compound that increases the strength or durationof the activity mediated by a histamine receptor, including the H3receptor or its variants.

“Antagonist” refers to a compound that decreases the strength orduration of the activity mediated by a histamine receptor, including theH3 receptor or its variants.

“Inverse agonist” refers to a compound that blocks agonist-inducedactivation of a histamine receptor and blocks constitutive or nativereceptor activity by preferentially binding to the inactive conformationof a G-protein coupled receptor such as a histamine receptor.

“Antibody” includes both monoclonal and polyclonal antibodies andincludes single chain or multi-chain antibodies, for example, antibodyfragments such as Fv, F(ab)₂, Fab, and also bispecific antibodies, scFv,humanized, chimeric, and human antibodies, including human antibodiesfrom transgenic animals with human immunoglobulin genes.

“Polynucleotide” includes single or double stranded nucleic acidpolymers of any length, both DNA and RNA and mixed polymers.

“Antisense nucleic acid” refers to a nucleic acid sequence, of anylength, that is complementary to the coding strand (sense strand) of thecoding region of the H3 receptor variants.

“H3 receptor variant” or “receptor variant” refers to an H3g, H3h, H3i,H3j, H3k or H3l polypeptide as desrcibed herein (see, e.g., SEQ ID NO:14, 16, 18, 20, 22 or 24).

“H3 wild type receptor” refers to an H3 receptor protein as described inU.S. Pat. No. 6,136,559, see also Lovenberg et al. (1999) Mol.Pharmacol. 55:1101 and WO 00/20011 (PCT/US98/21090), the amino acidsequence of which is set forth as SEQ ID NO:2.

Polypeptides

The invention provides isolated polypeptides comprising SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24,or polypeptides of at least 8 contiguous amino acid residues of any ofthe aforenamed SEQ ID NOs, wherein the polypeptides include at least theproline that is present at residue 6 (Pro6) and the leucine that ispresent at residue 7 (Leu7) in any of the aforementioned SEQ ID NOs.Such polypeptides will preferably comprise at least 10 contiguous aminoacid residues, more preferably at least 12 contiguous amino acidresidues, most preferably at least 15, 20 or 30 contiguous amino acidresidues of the sequence of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.

The polypeptides of the present invention include various modified formsof the H3 receptor polypeptides of SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, including fragments,analogs and variants thereof. The modifications that occur in apolypeptide often will be a function of how it is made. For polypeptidesmade by expressing a cloned gene in a host, for instance, the nature andextent of the modifications in large part will be determined by the hostcell's post-translational modification capacity and the modificationsignals present in the polypeptide amino acid sequence. For instance, asis well known, glycosylation often does not occur in bacterial hostssuch as E. coli. Accordingly, when glycosylation is desired, apolypeptide should be expressed in a glycosylating host, generally aeukaryotic cell. Insect cells often carry out the samepost-translational glycosylations as mammalian cells do and, for thisreason, insect cell expression systems have been developed to expressefficiently mammalian proteins having the native patterns ofglycosylation, inter alia. The polypeptides may be glycosylated invitro. Particularly preferred methods for producing glycosylationmodifications include exposing the mammalian histamine receptors toglycosylating enzymes derived from cells that normally carry out suchprocessing, such as mammalian glycosylation enzymes. Alternatively,deglycosylation enzymes can be used to remove carbohydrates attachedduring production in eukaryotic expression systems. Similarconsiderations apply to other modifications.

It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications of amino acid residues may include but are not limited toaliphatic esters or amides of the carboxyl terminus or of residuescontaining carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino-terminalamino acid or amino-group containing residues, e.g., lysine or arginine.Such modifications may also include but are not limited toglycosylation, pegylation, formylation and biotinylation.

The polypeptides of the invention can comprise subsequences, includingfragments, of the complete sequence of any of the H3 receptor variants.The subsequences, including fragments, will comprise the amino acid(s)flanking the N-terminal deletion site and thus the fragments are uniqueto the H3 receptor variants of the present invention. The fragments canbe produced by proteolytic cleavage of an intact H3 receptor variant,they can also be made by chemical synthesis or by the application ofrecombinant DNA technology and are not limited to polypeptidesdelineated by proteolytic cleavage sites. The polypeptides, either aloneor cross-linked or conjugated to a carrier molecule to render them moreimmunogenic, are useful as antigens to elicit the production ofantibodies. The antibodies can be used, e.g., in immunoassays of theintact H3 receptor variants, for immunoaffinity purification, etc. Thepolypeptides are also useful in the methods of the present invention forscreening antibody libraries or for identifying agonists, antagonists,or inverse agonists of the H3 receptor and receptor variants.

The polypeptides of the invention also include analogs of the H3receptor variants. The term “analog” means a H3 receptor variant of theinvention that has been modified by deletion, addition, modification orsubstitution of one or more amino acid residues in the H3 receptorvariant, with the proviso that the analog does not include the wildtypeH3 receptor. In addition, the term analog encompasses allelic andpolymorphic variants, and also muteins and fusion proteins whichcomprise all or a significant part of such a mammalian histamine H3receptor variant, e.g., covalently linked via a side-chain group orterminal residue to a different protein, polypeptide or moiety (fusionpartner). Where the analog contains one or more amino acid substitutionwith respect to the H3 receptor variant, the substitution is preferablya conservative substitution. By “conservative substitution” is intendeda replacement of the naturally occuring amino acid residue with aphysically or chemically similar residue, such as Gly/Ala, Asp/Glu,Val/Ile/Leu, Lys/Arg, Asn/Gln and Phe/Trp/Tyr as is well known in theprotein chemistry field. Analogs having such conservative substitutionstypically retain substantial histamine binding activity. Other analogs,which have non-conservative substitutions such as Asn/Glu, Val/Tyr andHis/Glu, may substantially lack such activity. Nevertheless, suchanalogs are useful because they can be used as antigens to elicitproduction of antibodies in an immunologically competent host or can beused to screen an antibody library. Because these analogs retain many ofthe epitopes (antigenic determinants) of the naturally occuring receptorvariants from which they are derived, many antibodies produced againstthem can also bind to the active-conformation or inactive (e.g.,denatured) forms of the naturally occurring receptors. Accordingly, suchantibodies can also be used, e.g., for the immunopurification orimmunoassay of the naturally occuring receptors and receptor variants.Other analogs include peptides having incorporation (substitution orinsertion) of unnatural or non-genetically encoded amino acid residues,or phosphorylated amino acid residues such as phosphotyrosine,phosphoserine or phosphothreonine residues. Guidance in determiningwhich nucelotides or amino acids may be substituted, inserted or deletedwithout altering or abolishing biological or immunological activity maybe found using computer programs that are well known in the art, forexample, DNASTAR software. “Substantial retention” of ligand bindingactivity by the foregoing analogs of the histamine receptor variantsmeans retention of at least about 50% of the histamine binding activityand/or specificity of the corresponding H3 receptor variant. Preferablythe analogs will retain at least about 75%, more preferably at leastabout 80%, and most preferably at least about 90% of the histaminebinding activity and/or specificity of the corresponding H3 receptorvariant.

Preferred analog embodiments further include those comprising apolypeptide having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 99%identity to a polypeptide reference sequence of SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24,wherein the polypeptide includes the proline at residue 6 and theleucine at residue 7 in any of the above-mentioned SEQ ID NOs.“Identity”, as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide. “Identity” and “similarity” can be readily calculated byknown methods, including but not limited to those described in(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12 (1):387(1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J.Mol. Biol. 215:403–410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol.215:403–410 (1990). The well-known Smith Waterman algorithm may also beused to determine identity. Preferred parameters for polypeptidesequence comparison include the following: 1) Algorithm: Needleman andWunsch, J. Mol. Biol. 48:443–453 (1970) Comparison matrix; 2) BLOSSUM62from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915–10919(1992) using a Gap Penalty: 12 and a Gap Length Penalty: 4; 3) Manualcomparison.

The analog polypeptide sequence will include at least one up to acertain integer number of amino acid alterations as compared to thesequence of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQID NO:22, or SEQ ID NO:24, wherein said alterations are selected fromthe group consisting of at least one amino acid deletion, substitution,including conservative and non-conservative substitution, or insertion,wherein the substituted or inserted amino acids may be naturallyoccurring or non-naturally occurring amino acids, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in thereference sequence by the integer defining the percent identity dividedby 100 and then subtracting that product from said total number of aminoacids in the reference sequence, or: n_(a)=x_(a)−(x_(a)y), wherein n_(a)is the number of amino acid alterations, x_(a) is the total number ofamino acids in the reference sequence, y is 0.50 for 50%, 0.60 for 60%,0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,0.97 for 97% or 0.99 for 99%, and is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).Preferably, the amino acids that flank the deletion site (that is, Pro6and Leu7 in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQID NO:22, or SEQ ID NO:24) will not be altered in the analog.

Analogs of the mammalian histamine receptor variants can be prepared bychemical synthesis or by recombinant methods using site-directedmutagenesis (Gillman et al., Gene 8:81 (1979); Roberts et al., Nature,328:731 (1987) or Innis (Ed.), 1990, PCR Protocols: A Guide to Methodsand Applications, Academic Press, New York, N.Y.) or the polymerasechain reaction method (PCR; Saiki et al., Science 239:487 (1988)), asexemplified by Daugherty et al. (Nucleic Acids Res. 19:2471 (1991)) tomodify nucleic acids encoding the receptor variants. Adding epitope tagsfor purification or detection of recombinant or synthetic products iscontemplated.

Polynucleotides

The present invention provides isolated polynucleotides that encodepolypeptides having the amino acid sequence of the histamine H3 receptorvariants, encode subsequences, including fragments, of histamine H3receptor variants, or encode genetically-encoded analogs of theforegoing. The polynucleotides of the present invention encode at least8 contiguous amino acids of the polypeptides having amino acid sequencesSEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, orSEQ ID NO:24. The at least 8 contiguous amino acids encoded willgenerally include the Pro6 and the Leu7 residues. The polynucleotides ofthe present invention include the coding strands as well as thecomplementary sequences, including polynucleotides useful as antisensemolecules. Also provided are recombinant vectors, particularlyexpression vectors, comprising the isolated polynucleotides, and hostcells containing the vectors. The polynucleotides of the invention areuseful, inter alia, for recombinant production of the H3 receptorvariants, as probes for detection of H3 receptor variant RNA or gene,restriction digestion analysis, as primers for PCR, or other enzymaticamplification method.

“Isolated polynucleotide” refers to a single-stranded or double-strandednucleic acid such as an RNA or DNA molecule, or a mixed polymer, whichis substantially separated from other components that are normally foundin cells or in recombinant DNA expression systems. These componentsinclude but are not limited to ribosomes, polymerases, serum components,and flanking genomic sequences. The term thus embraces a nucleic acidthat has been removed from its naturally occurring environment, andincludes recombinant or cloned DNA isolates and chemically synthesizedanalogs or analogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule. Noparticular size or size range of polymer is intended by use of the term“polynucleotide.”

An isolated polynucleotide will generally be a homogeneous compositionof molecules having identical nucleotide sequences but may, in someembodiments, contain minor sequence heterogeneity. Such heterogeneity istypically found at the ends of nucleic acid coding sequences or inregions not critical to a desired biological function or activity.

A “recombinant nucleic acid” is defined either by its method ofproduction or structure. Some recombinant nucleic acids are thus made bythe use of recombinant DNA techniques which involve human intervention,either in manipulation or selection. Others are made by fusing twofragments that are not naturally contiguous to each other. An isolatedpolynucleotide includes recombinant nucleic acid. Engineered vectors areencompassed, as well as nucleic acids comprising sequences derived usingany synthetic oligonucleotide process.

As mentioned above, the present invention provides isolatedpolynucleotides encoding the histamine H3 receptor variants.Polynucleotides comprising the nucleotide sequence encoding thepolypeptides having the amino acid sequence SEQ ID NO:14, SEQ ID NO:16,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24 are includedamong the polynucleotides of the present invention. SEQ ID NO: 13represents the sequence of a cDNA coding for the histamine H3 receptorvariant H3g. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, andSEQ ID NO:23 represent the coding sequences for H3 receptor variantsH3i, H3h, H3j, H3k, and H3l, respectively. As would be well understoodby one of ordinary skill in the art, because of the degeneracy of thegenetic code, there are many other polynucleotides other than thosehaving the sequences of the particular SEQ ID NOs above that can encodethe H3 receptor variants. For example, a codon found in the codingregion of the sequences set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 may be replaced witha degenerate codon encoding the same amino acid residue. All of thesedegenerate polynucleotides are included among the polynucleotides of thepresent invention.

The polynucleotides of the present invention also include thosepolynucleotides encoding fragments of the H3 receptor variants, as wellas those encoding genetically encoded analogs of the H3 receptorvariants. By “genetically encoded” analogs is intended H3 receptorvariant analog in which all of the amino acids comprising the analog areselected from the twenty genetically encoded amino acids.

For example, a wildtype codon may be replaced with a codon encoding aconservative substitution, while at the same time introducing orremoving a nucleic acid sequence recognition site, for example, arestriction site. Similarly, nucleic acid segments encoding desiredfunctions may be fused to generate a single genetic entity encoding adesired combination of functions not found together in nature. Althoughrestriction enzyme recognition sites are often the targets of suchartificial manipulations, other site-specific targets, e.g., promoters,DNA replication sites, regulation sequences, control sequences, or otheruseful features may be incorporated by design. Sequences encodingepitope tags for detection or purification as described above may alsobe incorporated. The polynucleotides encoding fragments of the H3receptor variants are generally at least 24 contiguous nucleotides inlength, preferably at least 30 contiguous nucleotides, more preferablyat least 36, most preferably at least 45, 60 or 90 contiguousnucleotides of the sequence of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, or degenerate versionsthereof. By “degenerate version” is intended any polynucleotide sequencethat encodes the same polypeptide sequence.

This invention further encompasses recombinant DNA molecules havingsequences that are homologous to those described herein. The nucleicacids of the invention may be operably linked to DNA segments thatcontrol transcription, translation, and DNA replication.

Preferred homologous polynucleotide embodiments include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 99% identity to the reference sequenceof SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,or SEQ ID NO:23, that includes a codon for proline at amino acid residue6 and a codon for leucine at amino acid 7. “Identity” is as definedpreviously. Preferred parameters for polynucleotide comparison includethe following: 1) Algorithm: Needleman and Wunsch, J. Mol. Biol.48:443–453 (1970) with a Comparison matrix: matches=+10, mismatch=0, GapPenalty: 50, and Gap Length Penalty: 3. A suitable program is availableas the Gap program from Genetics Computer Group, located in Madison,Wis. The homologous polynucleotide will include at least one up to acertain integer number of nucleotide alterations as compared to thesequence of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQID NO:21, or SEQ ID NO:23, wherein said alterations are selected fromthe group consisting of at least one nucleotide deletion, substitution,including transition and transversion, or insertion, and wherein saidalterations may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among the nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of nucleotide alterations isdetermined by multiplying the total number of nucleotides in SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ IDNO:23 by the integer defining the percent identity divided by 100 andthen subtracting that product from said total number of nucleotides inSEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, orSEQ ID NO:23, or n_(n)=x_(n)−(x_(n) y), wherein n_(n) is the number ofnucleotide alterations, x_(n) is the number of nucleotides in thereference sequence, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 0.99for 99%, and is the symbol for the multiplication operator, and whereinany non-integer product of x_(n) and y is rounded down to the nearestinteger prior to subtracting it from x_(n). Alterations of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24 maycreate nonsense, missense or frameshift mutations in this codingsequence and thereby alter the polypeptide encoded by the polynucleotidefollowing such alterations.

“Homologous nucleic acid sequences” or “homologous polynucleotides” arethose which when aligned and compared exhibit significant % identitiesas described above. Standards for homology in nucleic acids are eithermeasures for homology generally used in the art by sequence comparisonor based upon hybridization conditions, which are described in greaterdetail below. Substantial homology also exists when one sequence willhybridize under selective hybridization conditions to another.Typically, selective hybridization will occur when there is at leastabout 55% identity over a stretch of at least about 30 nucleotides,preferably at least about 65% identity over a stretch of at least about25 nucleotides, more preferably at least about 75%, and most preferablyat least about 90% identity over about 20 nucleotides. See, e.g.,Kanehisa, Nucleic Acids Res. 12:203 (1984).

The lengths of such homology comparisons may encompass longer stretchesand in certain embodiments may cover a sequence of at least about 17,preferably at least about 25, more preferably at least about 50, andmost preferably at least about 75 nucleotide residues.

Stringency of conditions employed in hybridizations to establishhomology are dependent upon factors such as salt concentration,temperature, the presence of organic solvents, and other parameters.Stringent temperature conditions usually include temperatures in excessof about 30° C., often in excess of about 37° C., typically in excess ofabout 45° C., preferably in excess of about 55° C., more preferably inexcess of about 65° C., and most preferably in excess of about 70° C.Stringent salt conditions will ordinarily be less than about 1000 mM,usually less than about 500 mM, more usually less than about 400 mM,preferably less than about 300 mM, more preferably less than about 200mM, and most preferably less than about 150 mM. For example, saltconcentrations of 100, 50 and 20 mM are used. The combination of theforegoing parameters, however, is more important than the measure of anysingle parameter. See, e.g., Wetmur et al., J. Mol. Biol. 31:349 (1968).

A further indication that two nucleic acid sequences that encodepolypeptides are substantially identical is that the polypeptide encodedby the first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid.

Polynucleotides encoding the H3 receptor variants, including fragmentsor analogs thereof, can be prepared by standard methods. For example,DNA can be chemically synthesized using, e.g., the phosphoramidite solidsupport method of Matteucci et al. (J. Am. Chem. Soc. 103:3185 (1981)),the method of Yoo et al. (J. Biol. Chem. 764:17078 (1989)), or otherwell known methods. This can be done by sequentially linking a series ofoligonucleotide cassettes comprising pairs of syntheticoligonucleotides. Alternatively, the polynucleotides can be synthesizedenzymatically, for example in a PCR reaction, or can be producedrecombinantly by amplifying a vector containing the H3 receptor variantcoding region, for example SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, or SEQ ID NO:23. In particular, enzymaticsynthesis is a convenient techniques for producing polynucleotides thatare fragments of the full length coding regions.

Moreover, nucleic acids encoding the H3 receptor variants can readily bemodified by nucleotide substitutions, nucleotide deletions, nucleotideinsertions, and inversions of nucleotide stretches. Such modificationsresult in novel DNA sequences that encode antigens having immunogenic orantigenic activity in common with the naturally occurring H3 receptorvariants. These modified sequences can be used to produce wild type ormutant receptors, or to enhance expression in a recombinant DNA system.

Insertion of the polynucleotides encoding the histamine H3 receptorvariants into a vector is easily accomplished when the termini of boththe receptor variant polynucleotide and the vector comprise compatiblerestriction sites. If this cannot be done, it may be necessary to modifythe termini of the polynucleotide and/or vector by digesting backsingle-stranded DNA overhangs generated by restriction endonucleasecleavage to produce blunt ends, or to achieve the same result by fillingin the single-stranded termini with an appropriate DNA polymerase.Alternatively, desired sites may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated by the use ofthe polymerase chain reaction (PCR). See, e.g., Saiki et al., Science239:487 (1988). The cleaved vector and the DNA fragments may also bemodified if required by homopolymeric tailing.

Recombinant expression vectors used in this invention are typicallyeither self-replicating DNA or RNA constructs, or an integrated plasmidor retrovirally encoded DNA, comprising a polynucleotide encoding one ofthe H3 receptor variants, or fragments or analogs thereof, usuallyoperably linked to suitable genetic control elements that are capable ofregulating expression of the nucleic acids in compatible host cells.Genetic control elements may include a prokaryotic promoter system or aeukaryotic promoter expression control system, and typically include atranscriptional promoter, an optional operator to control the onset oftranscription, transcription enhancers to elevate the level of mRNAexpression, a sequence that encodes a suitable ribosome binding site,and sequences that terminate transcription and translation. Expressionvectors also may contain an origin of replication that allows the vectorto replicate independently of the host cell, or as in the case ofrecombinant retroviral vectors or the integrated plasmid vector,replicate with the host genome.

Vectors that could be used in this invention include microbial plasmids,viruses, bacteriophage, integratable DNA fragments, and other vehiclesthat may facilitate integration of the nucleic acids into the genome ofthe host. Plasmids are the most commonly used form of vector but allother forms of vectors which serve an equivalent function and which are,or become, known in the art are suitable for use herein. See, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 andSupplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth,Boston, Mass.

Expression of nucleic acids encoding the histamine receptors, andreceptor variants, and fragments and analogs thereof, of this inventioncan be carried out by conventional methods in either prokaryotic oreukaryotic cells. Although strains of E. coli are employed mostfrequently in prokaryotic systems, many other bacteria such as variousstrains of Pseudomonas and Bacillus are know in the art and can be usedas well. Prokaryotic expression control sequences typically used includepromoters, including those derived from the β-lactamase and lactosepromoter systems (Chang et al., (1977) Nature, 198:1056), the tryptophan(trp) promoter system (Goeddel et al., (1980) Nucleic Acids Res.8:4057), the lambda PL promoter system (Shimatake et al., (1981) Nature,292:128) and the tac promoter (De Boer et al., (1983) Proc. Natl. Acad.Sci. USA 292:128). Numerous expression vectors containing such controlsequences are known in the art and available commercially.

Suitable host cells for expressing nucleic acids encoding the H3receptor variants or fragments or analogs thereof include prokaryotesand higher eukaryotes. Prokaryotes include both gram negative andpositive organisms, e.g., E. coli and B. subtilis. Higher eukaryotesinclude established tissue culture cell lines from animal cells, both ofnon-mammalian origin, e.g., insect cells, and birds, and of mammalianorigin, e.g., human, primates, and rodents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express themammalian histamine receptor variants include but are not limited tothose containing the lac promoter (pUC-series); trp promoter(pBR322-tip); Ipp promoter (the pIN-series); lambda-pL or pR promoters(pOTS); or hybrid promoters such as ptac (pDR540). See Brosius et al.,“Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derivedPromoters”, in Rodriguez and Denhardt (eds.) Vectors: A Survey ofMolecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston,pp. 205–236.

Higher eukaryotic tissue culture cells are preferred hosts for therecombinant production of the H3 receptor variants or fragments oranalogs thereof. Although any higher eukaryotic tissue culture cell linemight be used, including insect baculovirus expression systems,mammalian cells are preferred. Transformation or transfection andpropagation of such cells have become a routine procedure. Examples ofuseful cell lines include HeLa cells, Chinese hamster ovary (CHO) celllines, baby rat kidney (BRK) cell lines, insect cell lines, bird celllines, and monkey (COS) cell lines.

Expression vectors for such cell lines usually include an origin ofreplication, a promoter, a translation initiation site, RNA splice sites(if genomic DNA is used), a polyadenylation site, and a transcriptiontermination site. These vectors also usually contain a selection gene oramplification gene. Suitable expression vectors may be plasmids,viruses, or retroviruses carrying promoters derived, e.g., from suchsources as adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pCR.RTM.3.1, pcDNA1, pCD (Okayama et al., (1985) Mol. Cell Biol.5:1136), pMClneo Poly-A (Thomas et al., (1987) Cell 51:503), pUC19,pREP8, pSVSPORT and derivatives thereof, and baculovirus vectors such aspAC 373 or pAC 610. Particularly useful vectors are those containing theorigin of replication oriP of Epstein Barr virus, e.g., pCEP4(Invitrogen).

Accordingly, one aspect of the present invention provides a method formaking a polypeptide having the amino acid sequence of an H3 histaminereceptor variant or a fragment or an analog thereof. In particular theinvention provides a method for making a polypeptide comprising theamino acid sequence of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, or SEQ ID NO:24 by culturing a host cell comprisinga recombinant expression vector, wherein the expression vector comprisesa polynucleotide comprising SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, under conditions suitablefor expression of the receptor polypeptide, and recovering the expressedpolypeptide from the host cell culture.

Purification of Polypeptides

The H3 receptor variant polypeptides, including fragments and analogsthereof, of this invention can be purified from the host cell culturedescribed above, or from any other cell or tissue sources, or from asynthetic milieu, by standard methods, including but not limited to saltor alcohol precipitation, preparative disc-gel electrophoresis,isoelectric focusing, high pressure liquid chromatography (HPLC),reversed-phase HPLC, gel filtration, cation and anion exchange andpartition chromatography, and countercurrent distribution. Suchpurification methods are well known in the art and are disclosed, e.g.,in Guide to Protein Purification, Methods in Enzymology, Vol. 182, M.Deutscher, Ed., 1990, Academic Press, New York, N.Y. More specificmethods applicable to purification of the histamine receptors aredescribed below.

Purification steps can be followed by carrying out assays for ligandbinding activity as described below. Particularly where a receptorvariant is being isolated from a cellular or tissue source, it ispreferable to include one or more inhibitors of proteolytic enzymes inthe assay system, such as phenylmethanesulfonyl fluoride (PMSF).

Antisense Polynucleotides

The present invention provide antisense polynucleotides that can beemployed to partially or totally eliminate expression of specific genesor specific mRNA splice variants. See, Helene and Toulme, 1990,Biochimica Biophys. Acta 1049:99; Pepin et al., 1991 Nature 355:725,Stout and Caskey, 1990, Somat. Cell Mol. Genet. 16:369; Munir et al.,1990, Somat. Cell Mol. Genet. 16:383.

Generally, antisense polynucleotides for the purposes of the inventionare complementary to parts of the sequence of the coding region of theH3 variants. Complementary antisense polynucleotides include antisenseRNA which can hybridize specifically to individual mRNA species andhinder or prevent transcription and/or RNA processing of the mRNAspecies and/or translation of the encoded polypeptide (Ching et al.,1989, Proc. Natl. Acad. Sci. USA 86:10006–10010; Broder et al., Ann.Int. Med. 113:604–618; Loreau et al., 1990, FEBS Letters 274:53–56;Holcenberg et al., WO91/11535; WO91/09865; WO91/04753; WO90/13641; andEP 386563). An antisense sequence is a polynucleotide sequence of atleast about 15 contiguous nucleotides in length, typically at least 20to 30 nucleotides in length, and preferably more than about 30nucleotides in length that is substantially complementary to a targetcoding region sequence, or sequences. At a minimum, the antisensepolynucleotides will comprise sequence that is complementary to thesequences encoding the amino acids at the deletion site, that is, Pro6and Leu7. In some embodiments, antisense sequences may havesubstitutions, additions, or deletions as compared to the complementarytarget sequence but as long as specific hybridization is retained, thepolynucleotide will generally function as an antisense inhibitor of geneexpression.

Antibodies

The present invention provides antibodies that specifically recognizethe H3 receptor variants. By “specifically recognize” is intended thatthe antibody binds preferentially to the H3g, H3h, H3i, H3j, H3k, or H3lvariant, for example, with a higher affinity than to the wild type H3receptor. In general, the antibodies of the invention will have at leasttwo-fold higher affinity for one of the H3 variants than for the wildtype H3 receptor. Preferably, the antibodies of the invention will haveat least 5-fold higher affinity for one of the H3 variants than for thewild type H3 receptor, more preferably the antibodies of the inventionwill have at least 10-fold higher or a 100-fold higher affinity for oneof the H3 variants than for the wild type H3 receptor. Antibodies thatspecifically recognize the H3 receptor variants of the present inventionmay recognize an epitope that is not present on the H3 wild typereceptor (for instance, an epitope that is present at the region encodedby the new splice junction in the variants) or an epitope that isrevealed due to a conformational change in the H3 receptor variantscompared to the H3 wild type receptor. H3 wild type receptors includethe H3 receptor as previously described by Lovenberg et al. (U.S. Pat.No. 6,136,559; WO 00/20011 (PCT/US98/21090)), the amino acid sequence ofwhich is set forth in SEQ ID NO:2.

Antigenic (i.e., immunogenic) polypeptides, including subsequences andfragments, of H3 receptor variants of this invention, which may or maynot have ligand binding activity, may be produced. Regardless of whetherthey bind histamine, such polypeptides, like the complete receptors, areuseful as antigens for preparing antibodies by standard methods that canbind to the complete receptor variants. Shorter subsequences based on orderived from the receptor variants, including fragments, can beconcatenated or attached to a carrier. Because it is well known in theart that epitopes generally contain at least about five, preferably atleast about 8, amino acid residues (Ohno et al., Proc. Natl. Acad. Sci.USA 82:2945 (1985)), fragments used for the production of antibodieswill generally be at least that size. Preferably, they will contain evenmore residues, as described above. Whether a given polypeptide isimmunogenic can readily be determined by routine experimentation.

Although it is generally not necessary when full length receptorvariants are used as antigens to elicit antibody production in animmunologically competent host, smaller antigenic fragments arepreferably first rendered more immunogenic by cross-linking orconcatenation, or by coupling to an immunogenic carrier molecule (i.e.,a macromolecule having the property of independently eliciting animmunological response in a host animal). Cross-linking or conjugationto a carrier molecule may be required because small polypeptidefragments sometimes act as haptens (molecules which are capable ofspecifically binding to an antibody but incapable of eliciting antibodyproduction, i.e., they are not immunogenic). Conjugation of suchfragments to an immunogenic carrier molecule renders them moreimmunogenic through what is commonly known as the “carrier effect”.

Suitable carrier molecules include, e.g., proteins and natural orsynthetic polymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides etc. Protein carrier molecules are especiallypreferred, including but not limited to keyhole limpet hemocyanin andmammalian serum proteins such as human or bovine gammaglobulin, human,bovine or rabbit serum albumin, or methylated or other derivatives ofsuch proteins. Other protein carriers will be apparent to those skilledin the art. Preferably, but not necessarily, the protein carrier will beforeign to the host animal in which antibodies against the fragments areto be elicited.

Covalent coupling to the carrier molecule can be achieved using methodswell known in the art, the exact choice of which will be dictated by thenature of the carrier molecule used. When the immunogenic carriermolecule is a protein, the fragments of the invention can be coupled,e.g., using water-soluble carbodiimides such as dicyclohexylcarbodiimideor glutaraldehyde.

Coupling agents such as these can also be used to cross-link thefragments to themselves without the use of a separate carrier molecule.Such cross-linking into aggregates can also increase immunogenicity.Immunogenicity can also be increased by the use of known adjuvants,alone or in combination with coupling or aggregation.

Suitable adjuvants for the vaccination of animals include but are notlimited to Adjuvant 65 (containing peanut oil, mannide monooleate andaluminum monostearate); Freund's complete or incomplete adjuvant;mineral gels such as aluminum hydroxide, aluminum phosphate and alum;surfactants such as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl)propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The polypeptides could also be administered following incorporation intoliposomes or other microcarriers.

Information concerning adjuvants and various aspects of immunoassays aredisclosed, e.g., in the series by P. Tijssen, Practice and Theory ofEnzyme Immunoassays, 3rd Edition, 1987, Elsevier, N.Y. Other usefulreferences covering methods for preparing polyclonal antisera includeMicrobiology, 1969, Hoeber Medical Division, Harper and Row;Landsteiner, Specificity of Serological Reactions, 1962, DoverPublications, New York, and Williams, et al., Methods in Immunology andImmunochemistry, Vol. 1, 1967, Academic Press, New York.

Serum produced from animals immunized using standard methods can be useddirectly, or the antibody fraction can be separated from the serum usingstandard methods such as plasmaphoresis or adsorption chromatographywith IgG-specific adsorbents such as immobilized Protein A.Alternatively, monoclonal antibodies can be prepared.

Hybridomas producing monoclonal antibodies against the H3 receptorvariants of the invention or antigenic fragments or analogs thereof areproduced by well-known techniques. Usually, the process involves thefusion of an immortalizing cell line with a B-lymphocyte that producesthe desired antibody. Alternatively, non-fusion techniques forgenerating immortal antibody-producing cell lines can be used, e.g.,virally-induced transformation (Casali et al., Science 234:476 (1986)).Immortalizing cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine, and human origin. Mostfrequently, rat or mouse myeloma cell lines are employed as a matter ofconvenience and availability.

Techniques for obtaining antibody-producing lymphocytes from mammalsinjected with antigens are well known, including from transgenic animalswith human immunoglobulin genes. Generally, peripheral blood lymphocytes(PBLs) are used if cells of human origin are employed, or spleen orlymph node cells are used from non-human mammalian sources. A hostanimal is injected with repeated dosages of the purified antigen (humancells are sensitized in vitro), and the animal is permitted to generatethe desired antibody-producing cells before they are harvested forfusion with the immortalizing cell line. Techniques for fusion are alsowell known in the art, and in general involve mixing the cells with afusing agent, such as polyethylene glycol.

Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. Those secreting thedesired antibody are selected using standard immunoassays, such asWestern blotting, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), or the like. Antibodies are recovered from themedium using standard protein purification techniques (Tijssen, Practiceand Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)).

Many references are available to provide guidance in applying the abovetechniques (Kohler et al., Hybridoma Techniques (Cold Spring HarborLaboratory, New York, 1980); Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal AntibodyTechnology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal HybridomaAntibodies: Techniques and Applications (CRC Press, Boca Raton, Fla.,1982)). Monoclonal antibodies can also be displayed, selected orproduced using well-known phage library systems. See, e.g., Huse, etal., Science 246:1275 (1989); Ward, et al., Nature, 341:544 (1989).

Antibodies thus produced, whether polyclonal or monoclonal, can be used,e.g., in an immobilized form bound to a solid support by well knownmethods, to purify the receptor variants by immunoaffinitychromatography.

Antibodies against the antigenic fragments can also be used, unlabeledor labeled by standard methods, as the basis for immunoassays of the H3receptor and the H3 receptor variants. The particular label used willdepend upon the type of immunoassay. Examples of labels that can be usedinclude but are not limited to radiolabels such as ³²P, ¹²⁵I, ³H and¹⁴C; fluorescent labels such as fluorescein and its derivatives,rhodamine and its derivatives, dansyl and umbelliferone;chemiluminescers such as luciferin and 2,3-dihydrophthalazinediones; andenzymes such as horseradish peroxidase, alkaline phosphatase, lysozymeand glucose-6-phosphate dehydrogenase.

The antibodies can be tagged with such labels by known methods. Forexample, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bisdiazotized benzadine and the like may be usedto tag the antibodies with fluorescent, cherm luminescent or enzymelabels. The general methods involved are well known in the art and aredescribed, e.g., in Immunoassay: A Practical Guide, 1987, Chan (Ed.),Academic Press, Inc., Orlando, Fla. Such immunoassays could be carriedout, for example, on fractions obtained during purification of thereceptor variants.

The antibodies of the present invention can also be used to identifyparticular cDNA clones expressing the H3 receptor variants in expressioncloning systems.

Neutralizing antibodies specific for the ligand-binding site of areceptor can also be used as antagonists (inhibitors) to block histaminebinding. Such neutralizing antibodies can readily be identified throughroutine experimentation, e.g., by using the radioligand binding assaydescribed infra. Antagonism of histamine activity can be accomplishedusing complete antibody molecules, single chain or multi-chain antibodymolecules, or well-known antigen binding fragments such as Fab, Fc,F(ab)₂, and Fv fragments.

Definitions of such fragments can be found, e.g., in Klein, Immunology(John Wiley, New York, 1982); Parham, Chapter 14, in Weir, ed.Immunochemistry, 4th Ed. (Blackwell Scientific Publishers, Oxford,1986). The use and generation of antibody fragments has also beendescribed, e.g.: Fab fragments [Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985)], Fv fragments [Hochman et al.,Biochemistry 12:1130 (1973); Sharon et al., Biochemistry 15:1591 (1976);Ehrlich et al., U.S. Pat. No. 4,355,023] and antibody half molecules(Auditore-Hargreaves, U.S. Pat. No. 4,470,925). Methods for makingrecombinant Fv fragments based on known antibody heavy and light chainvariable region sequences have further been described, e.g., by Moore etal. (U.S. Pat. No. 4,642,334) and by Pluckthun (Bio/Technology 9:545(1991)). Alternatively, they can be chemically synthesized by standardmethods.

Methods of Identifying Agonists, Inverse Agonists or Antagonists

The invention allows the discovery of selective agonists, inverseagonists or antagonists of the novel receptor variants that may beuseful in treatment and management of a variety of diseases includingcentral nervous system disorders, for example depression, anxiety,psychoses (for example schizophrenia), tardive dyskinesia, Parkinson'sdisease, obesity, hypertension, Tourette's syndrome, sexual dysfunction,gastrointestinal disorders, psychiatric disorders, sleep disorders,hypothalamic dysfunction, drug addiction, drug abuse, cognitivedisorders, Alzheimer's disease, senile dementia, narcolepsy, comasinduced by stroke, drugs or alcohol, obsessive-compulsive behavior,panic attacks, pain, social phobias, eating disorders and anorexia,appetite suppression for treatment of obesity, cardiovascular andcerebrovascular disorders, non-insulin dependent diabetes mellitus,hyperglycemia, constipation, arrhythmia, disorders of theneuroendrocrine system, stress, and spasticity, as well as acidsecretin, ulcers, airway constriction, asthma, allergy, inflammation,and prostate dysfunction. Thus, the H3 receptor variants of thisinvention can be employed in screening systems to identify agonists,antagonists or inverse agonists of the H3 receptor or the H3 receptorvariants. Essentially, these systems provide methods for bringingtogether a H3 receptor variant, an appropriate known ligand, includinghistamine itself, and a sample to be tested for the presence of ahistamine agonist, antagonist or inverse agonist.

At least two basic types of screening systems can be used, alabeled-ligand binding assay and a “functional” assay. A labeled ligandfor use in the binding assay can be obtained by labeling histamine or ahistamine agonist, inverse agonist or antagonist (i.e. a histaminesurrogate) with measurable group as described above in connection withthe labeling of antibodies. Various labeled forms of histamine areavailable commercially or can be generated using standard techniques. Inan example below, ³H-Nα-methyl histamine is used as the ligand.

Typically, a given amount of the H3 receptor variant the invention iscontacted with increasing amounts of a labeled ligand, such as histamineor ³H-Nα-methyl histamine, and the amount of the bound labeled ligand ismeasured after removing unbound labeled ligand by washing. As the amountof the labeled ligand is increased, a point is eventually reached atwhich all receptor variant binding sites are occupied or saturated.Specific receptor variant binding of the labeled ligand is abolished bya large excess of unlabeled ligand. Such histamine binding assays arewell known in the art and are described, e.g., in Tedford et al. (J.Pharmacol, Exp. Ther. 1995 275:598).

Preferably, an assay system is used in which non-specific binding of thelabeled ligand to the receptor is minimal. Non-specific binding istypically less than 50%, preferably less than 15%, and more preferablyless than 10% of the total binding of the labeled ligand.

In principle, a binding assay of the invention could be carried outusing a soluble receptor variant of the invention, e.g., followingproduction and refolding by standard methods from an E. coli expressionsystem, and the resulting receptor variant-labeled ligand complex couldbe precipitated, e.g., using an antibody against the receptor variant.The precipitate could then be washed and the amount of the bound labeledligand could be measured.

Preferably, however, the H3 receptor variant for use in the method willbe a membrane bound receptor variant. To prepare such membrane boundreceptor variants, a nucleic acid encoding one of the H3 receptorvariants of the invention is transfected into an appropriate host cell,whereby the receptor variant will become incorporated into the membraneof the cell. A membrane fraction can then be isolated from the cell andused as a source of the receptor variant for assay. Preferably, specificbinding of the labeled ligand to a membrane fraction from theuntransfected host cell will be negligible.

The binding assays of this invention can be used to identify bothhistamine agonists, inverse agonists and antagonists because suchcompounds will interfere with the binding of the labeled ligand to thereceptor variant.

In the basic binding assay, the method for identifying a histamineagonist, antagonist, or inverse agonist comprises:

(a) contacting a polypeptide comprising a receptor variant having anamino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, or a fragment oranalog thereof, in the presence of an amount of labeled ligand, such ashistamine or histamine surrogate, or antagonist with a sample to betested for the presence of a histamine agonist, inverse agonist;

(b) measuring the amount of labeled receptor ligand, such asNα-methylhistamine, bound to the receptor; and

(c) comparing the amount of labeled ligand receptor bound to thepolypeptide in the presence of said sample and in the absence of saidsample whereby a histamine agonist, inverse agonist or antagonist in thesample is identified by measuring substantially reduced binding of thelabeled receptor ligand to the polypeptide, compared to what would bemeasured in the absence of such agonist, inverse agonist or antagonist.

By “substantially reduced” is meant that the binding of histamine in thepresence of said sample is no more than 80%, preferably no more than70%, more preferably no more than 50%, most preferably no more than 40%,30%, 20% or 10%, of the binding of histamine in the absence of saidsample. Preferably, the polypeptide used to identify a histamineagonist, inverse agonist or antagonist for human therapeutic purposeshas an amino acid sequence SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQID NO:20, SEQ ID NO:22, or SEQ ID NO:24, or a fragment of any of theforegoing sequences.

Determination of whether a particular molecule inhibiting binding of thelabeled ligand to the receptor variant is an agonist, inverse agonist orantagonist is then determined in a second, functional assay. Thefunctionality of histamine agonists, inverse agonists or antagonistsidentified in the binding assay can be determined in cellular and animalmodels.

Functional Assays for Antagonists/Agonists/Inverse Agonists of HistamineReceptors

In cellular models, parameters for intracellular activities mediated byhistamine receptors can be monitored for antagonistic and/or agonisticand/or inverse agonistic activities. Such parameters include but are notlimited to intracellular second messenger pathways activated via the H3histamine receptors, changes in cell growth rate, secretion of hormones,etc., using published methods. Examples of such methods are, measurementof the effects of the ligands on receptor-mediated inhibition offorskolin-stimulated intracellular cAMP production (Parker et al., Mol.Brain Res. 34:179–189 (1995)), receptor-stimulated Ca²⁺ mobilization andmitogenic effects (Sethi et al., Cancer Res. 51:1674–1679 (1991)), andinositol phosphate production and MAP kinase induction (Wang et al.,Biochemistry 37:6711–17 (1998). The FLIPR method described in U.S. Pat.No. 6,204,017 is also suitable for measuring intracellular calciumrelease. Other suitable functional assays are described in Lovenberg etal. 1999 Mol. Pharmacol. 55:33 or Coge et al. 2001 Biochem. J. 355:279.

Agonists, inverse agonists or antagonists of histamine receptors mayalso be identified directly by using functional assays. An agonist,inverse agonist or antagonist may or may not directly inhibit histaminebinding to histamine receptors.

In addition to the methods described above, activities of an antagonistmay be measured in cellular models for altered intracellular cAMP orcalcium ion concentrations. Histamine-induced chemotaxis using culturedcells can also be utilized. Furthermore, models employing Xenopuslaevis, pigment dispersion/aggregation in melanophores, and aequorinassay in mammalian cells are suitable for this purpose. Methods usinganimals or animal tissues for such activities can also be employed.Histamine-stimulated neutrophil chemotaxis, enhancedneutrophil-endothelial interaction, neutrophil activation leading todegranulation and release of mediators, enzymes and superoxides,inflammatory pain, and increased cytokine production and transcriptionare examples of such methods.

In basic functional assay the method of the present invention thusprovides a method for identifying an agonist, inverse agonist orantagonist of a mammalian histamine receptor comprising:

1) contacting a polypeptide having the amino acid sequence of SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ IDNO:24, or a fragment or analog thereof, in the presence of an amount ofligand, such as histamine or a histamine surrogate with a sample to betested for the presence of a histamine agonist, inverse agonist orantagonist,

2) measuring at least one cellular function modulated by saidpolypeptide, and

3) comparing the at least one cellular function modulated by saidpolypeptide in the presence of said sample and in the absence of saidsample,

wherein a substantial change in the at least one cellular function inthe presence of said sample indicates the presence of a histamineagonist, antagonist, or inverse agonist. By “substantial change” isintended a change (either increase or decrease in activity or duration)of at least 10%. A substantial change will preferably be a change of atleast 20%, more preferably of at least 30%, 40%, 50% or 75%. It will beappreciated by one of ordinary skill in the art that the abovefunctional assay will be carried out with polypeptide provided in anenvironment in which the at least one cellular function can be measured,for instance in a whole cell or an isolated membrane.Agonists, Inverse Agonists and Antagonists Pharmaceutical Compositions

Agonists, inverse agonists or antagonist identified by the methods ofthe present invention are also included in the present invention as arepharmaceutical compositions comprising the identified agonists, inverseagonists or antagonists, and a pharmaceutically acceptable carrier.

The present invention, in one aspect, provides compounds, eitheragonists, inverse agonists or antagonists identified by the methodsdisclosed herein, which compounds are useful for the treatment ofdiseases, disorders and conditions associated with or modulated by theH3 histamine receptor. Some such conditions include those mentionedabove. Compounds (that is agonists, inverse agonists or antagonists)identified according to the methods disclosed herein may be used aloneat appropriate dosages defined by routine testing in order to obtainoptimal modulation (either activation or inhibition) of the humanhistamine H3 receptor variant or its activity while minimizing anypotential toxicity. In addition, co-administration or sequentialadministration of other agents may be desirable.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in methods of treatment of diseases and disorders associated withthe human H3 receptor and H3 receptor variant. The compositionscontaining compounds identified according to this invention as theactive ingredient for use in the modulation of human histamine H3receptor variants can be administered in a wide variety of therapeuticdosage forms in conventional vehicles for administration. U.S. Pat. Nos.5,380,858, 5,486,526, 5,633,382, 5,639,775, 5,652,258, 5,990,317,6,008,240, 6,072,057, and 6,166,060, which are incorporated herein byreference, describe the preparation of pharmaceutical compositionscomprising H3 receptor antagonists and administration thereof. Similartechniques are suitable for the compounds of the present invention. Forexample, the compounds or modulators can be administered in such oraldosage forms as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, solutions, suspensions, syrups and emulsions, or byinjection. Likewise, they may also be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, or intramuscular form, all using forms well known tothose of ordinary skill in the pharmaceutical arts. An effective butnon-toxic amount of the compound desired can be employed as a humanhistamine H3 receptor variant modulating agent (i.e, agonist,antagonist, or inverse agonist).

The daily dosage of the compounds may be varied over a wide range from0.01 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 1000 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. The dosages of the human histamine H3 receptor variantmodulators are adjusted when combined to achieve desired effects. On theother hand, dosages of these various agents may be independentlyoptimized and combined to achieve a synergistic result wherein thepathology is reduced more than it would be if either agent were usedalone.

Advantageously, compounds of the present invention may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three or four times daily. Furthermore, compoundsor modulators for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles, or viatransdermal routes, using those forms of transdermal skin patches wellknown to those of ordinary skill in that art. To be administered in theform of a transdermal delivery system, the dosage administration will,of course, be continuous rather than intermittent throughout the dosageregimen. For combination treatment with more than one active agent,where the active agents are in separate dosage formulations, the activeagents can be administered concurrently, or they each can beadministered at separately staggered times.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularcompound thereof employed. A physician or veterinarian of ordinary skillcan readily determine and prescribe the effective amount of the drugrequired to prevent, counter or arrest the progress of the condition.Optimal precision in achieving concentrations of drug within the rangethat yields efficacy without toxicity requires a regimen based on thekinetics of the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug.

In the methods of treatment of the present invention, the compoundsherein described in detail can form the active ingredient, and aretypically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents which may be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations which generally containsuitable preservatives are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eolis, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds or modulators of the presentinvention may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinyl-pyrrolidone, pyrancopolymer, polyhydroxypropylmethacrylamidephenyl,polyhydroxy-ethylaspartamidephenyl, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds ormodulators of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

For oral administration, the compounds may be administered in capsule,tablet, or bolus form or alternatively they can be mixed in the animalsfeed. The capsules, tablets, and boluses are comprised of the activeingredient in combination with an appropriate carrier vehicle such asstarch, talc, magnesium stearate, or di-calcium phosphate. These unitdosage forms are prepared by intimately mixing the active ingredientwith suitable finely-powdered inert ingredients including diluents,fillers, disintegrating agents, and/or binders such that a uniformmixture is obtained. An inert ingredient is one that will not react withthe compounds or modulators and which is non-toxic to the animal beingtreated. Suitable inert ingredients include starch, lactose, talc,magnesium stearate, vegetable gums and oils, and the like. Theseformulations may contain a widely variable amount of the active andinactive ingredients depending on numerous factors such as the size andtype of the animal species to be treated and the type and severity ofthe infection. The active ingredient may also be administered as anadditive to the feed by simply mixing the compound with the feedstuff orby applying the compound to the surface of the feed. Alternatively theactive ingredient may be mixed with an inert carrier and the resultingcomposition may then either be mixed with the feed or fed directly tothe animal. Suitable inert carriers include corn meal, citrus meal,fermentation residues, soya grits, dried grains and the like. The activeingredients are intimately mixed with these inert carriers by grinding,stirring, milling, or tumbling such that is the final compositioncontains from 0.001 to 5% by weight of the active ingredient.

The compounds may alternatively be administered parenterally viainjection of a formulation consisting of the active ingredient dissolvedin an inert liquid carrier. Injection may be either intramuscular,intraruminal, intratracheal, or subcutaneous. The injectable formulationconsists of the active ingredient mixed with an appropriate inert liquidcarrier. Acceptable liquid carriers include the vegetable oils such aspeanut oil, cotton seed oil, sesame oil and the like as well as organicsolvents such as solketal, glycerol formal and the like. As analternative, aqueous parenteral formulations may also be used. Thevegetable oils are the preferred liquid carriers. The formulations areprepared by dissolving or suspending the active ingredient in the liquidcarrier such that the final formulation contains from 0.005 to 10% byweight of the active ingredient.

Topical application of the compounds or modulators is possible throughthe use of a liquid drench or a shampoo containing the instant compoundsor modulators as an aqueous solution or suspension. These formulationsgenerally contain a suspending agent such as bentonite and normally willalso contain an antifoaming agent. Formulations containing from 0.005 to10% by weight of the active ingredient are acceptable. Preferredformulations are those containing from 0.01 to 5% by weight of theinstant compounds.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLES

Cloning of H3g and H3h Receptor Variants

Preparation of cDNA:

Poly A+ mRNA from human brain was obtained from Clontech (1020 EastMeadow Circle, Palo Alto, Calif. 94303-4230 USA) (Cat. 6548-1). The polyA+ mRNA was reverse transcribed using the Superscript II reversetranscription kit (Life Technologies, Cat. No.: 18064014) using randomhexamer primers (Roche Molecular Biochemicals, Indianapolis, Ind., Cat.No. 1034731). 50 ng of Poly A+ was used as the template. Transcriptionwas allowed to proceed at room temperature for 5 min., followed by 45min. at 50° C.

Molecular Cloning

The H3 Histamine sequences were cloned in two fragments by PCR from thehuman brain cDNA. The sequences for the primers are indicated below. Forthe N-terminus the following primers were used: HR352 and H₃NHE3. Theprimer H₃NHE3 contained an internal Nhe-1 restriction site which couldbe used for receptor assembly. This site was engineered into the H3sequence without disrupting the coding sequence of the receptor. Toclone the C-terminus of the H3 receptor the following primers were used:H3NHE5 and HR33. The PCR amplification of the H3 fragments was carriedout using the Expand mixture of DNA polymerases (Roche MolecularBiochemicals, Cat. No. 1732650). 5 uL of a 50 uL cDNA reaction was usedin PCR amplification using the following PCR conditions: (94° C., 2min.; [94° C., 40 sec., 60° C., 40 sec., 72° C., 1 min. 30 sec.] for 10cycles, [94° C., 40 sec., 60° C., 40 sec., 72° C., 1 min. 30 sec+20 secadded per cycle] for 25 cycles, 72° C. for 7 min., 4° C. hold. PCRamplification products were analyzed on a 1% agarose gel and revealed asingle band for the N-terminus of H3 which was 108 bases shorter thanthe previously published H3 sequence, resulting in a protein that was 36amino acids shorter than wild type H3. The PCR of the C-terminusresulted in two fragments which were 797 and 557 in length, the shortercontaining a 240 base pair (80 residue) deletion in the region encodingintracellular loop three. The PCR products were cloned into the vectorpAMP-1 (Life Technologies, Cat. No. 18381012), using the UDG cloningsystem. The engineered Nhe I site was used to assemble full-lengthconstructs by joining the N-terminal and C-terminal fragments.

In order to get the wild-type form of H3 (H3a) the deleted 108 basenucleotide sequence in our H₃N-terminus was reintroduced. This wasaccomplished by inserting the bases by seven rounds of PCRamplification. The seven rounds of PCR amplification were carried outwith the following primer sets: Round 1, H3EXT1+H3NHE3; Round 2,H3EXT2+H3NHE3; Round 3, H3EXT3+H3NHE3; Round 4, H3EXT4+H3NHE3; Round 5,H3EXT5+H3NHE3; Round 6, H3EXT6+H3NHE3; Round 7, HR352+H3NHE3. The PCRconditions used to amplify these sequences were as follows: (94° C., 2min.; [94° C., 40 sec., 60° C., 40 sec., 72° C., 1 min. 30 sec.] for 35cycles, 72° C. for 7 min., 4° C. hold). In all cases, the 1 μL ofamplification product from one round of PCR was diluted 1/10 and thenused as the template for the next round of amplification. The finalproduct was subcloned into p-AMP-1 as previously described. Theengineered Nhe-I site was again used for construct assembly.

The nucleotide sequences of the H3g and H3h variants are shown in SEQ IDNO: 13 and 17, respectively. The corresponding amino acid sequences areshown in SEQ ID NO: 14 and 18, respectively. The nucleotide sequence ofthe wild type H3 receptor (the H3a construct described above) is shownin SEQ ID NO:1, the protein sequence of the H3 wild type receptor isshown in SEQ ID NO:2. The nucleotide sequence of H3 variant having the240 base deletion in the C-terminal encoding region and the N-terminalregion from the wild type H3 receptor was designated H3b and thenucleotide sequence is shown in SEQ ID NO: 3, amino acid sequence in SEQID NO:4. The nucleotide and amino acid sequences of the H3 receptorvariants described in Coge et al. (2001, supra) are shown forcomparison. SEQ ID NO:5 and SEQ ID NO:6 show, respectively, thenucleotide and amino acid sequences of the H_(3(ΔTM2, 431aa)) variant.SEQ ID NO: 7 and SEQ ID NO:8 show, respectively, the nucleotide andamino acid sequences of the H_(3(Δi3, 415aa)). SEQ ID NO: 9 and SEQ IDNO:10 show, respectively, the nucleotide and amino acid sequences of theH_(3(Δi3, 329aa)). SEQ ID NO: 11 and SEQ ID NO:12 show, respectively,the nucleotide and amino acid sequences of the H_(3(ΔTM5+Δi3, 326aa)).

Transient Expression of H3 Histamine Receptor Variants in COS-7 Cellsand Determination of Histamine Binding

The full length wild type receptor (H3a) and receptor variants H3g andH3h, as well as the variant H3b (which contains the 240 base, 80 aminoacid deletion in intracellular loop 3 (i3) but not the N-terminal 108base, 36 amino acid deletion), were cloned in pCDNA3.0 eukaryoticexpression vector for expression in COS-7 cells. The COS-7 cells grownin culture chambers were transfected with 2 μg of plasmid DNA containingone of the H3 receptor or receptor variants with superfect (Qiagen)transfection reagent.

Binding of Histamine to receptor or receptor variant was determined byBODIPY-L-Histamine or Fluorescein Histamine (Molecular Probes). Briefly,the transfected cells were washed with PBS and 1 μM ofBODIPY-L-Histamine or Fluorescein Histamine in PBS was added to thecells and incubated at 37° C. for 30 min. Unbound fluorescent labeledhistamine was washed in cold PBS and the slides were examinedimmediately under a fluorescent microscope. Cells transfected with theH3g, H3h and H3b variants showed histamine binding comparable to thecell transfected with the H3a wild type receptor.

Primers used to Clone H3 Histamine Receptors: For N-terminus: PrimerHR352 (5′) (SEQ ID NO:25) 5′-CUACUACUACUAATGGAGCGCGCGCCGCCC-3′ PrimerH3NHE3 (3′) (SEQ ID NO:26) 5′-CAUCAUCAUCAUGCTAGCCGTGATGAGGAAGTACCAG ForC-terminus Primer H3NHE5 (5′) (SEQ ID NO:27)5′-CUACUACUACUAGCTAGCACCCTGGAGTTCTTTACGCCC Primer HR33 (3′) (SEQ IDNO:28) 5′-CAUCAUCAUCAUGGCCAGATGCCCAGGAGACC For Extension of theshortened H3 N-terminus the following primers were used Primer H3EXT1(5′) (SEQ ID NO:29) 5′-CTCATCGTGGCCACGGTGC-3′ Primer H3EXT2 (5′) (SEQ IDNO:30) 5′GGTGCTGGCCGCGCTCATGGCGCTGCTCATCGTGGCCACGGTGC-3′ Primer H3EXT3(5′) (SEQ ID NO:31) 5′GCGGCTTCTCGGCAGCCTGGACCGCGGTGCTGGCCGCGCTCATGG-3′Primer H3EXT4 (5′) (SEQ ID NO:32)5′GATGCGGCGGCGGCGGGCGGGGCGCGCGGCTTCTCGGCAGCCTG-3′ Primer H3EXT5 (5′)(SEQ ID NO:33) 5′GAACGCTTCGGGGGCGCTGGCGGGCGATGCGGCGGCGGCGG-3 PrimerH3EXT6 (5′) (SEQ ID NO:34) 5′AGCGCGCGCCGCCCGACGGGCCGCTGAACGCTTCGGGGGCG-3Membrane Preparations

Cell transfected with the H3 receptor variant plasmid alone, orco-transfected with G-protein plasmid(s) are harvested by incubating in5 mM EDTA/phosphate-buffered saline followed by repeated pipetting. Thecells are centrifuged for 5 min at 1000 g. The EDTA/PBS is decanted, andan equal volume of ice-cold 50 mM Tris-HCl, pH 7.5, is added and cellsare broken up with a Polytron homogenizer (PT-10 tip, setting 5, 30s).Nuclei and unbroken cells are sedimented at 1,000 g for 10 min and thenthe supernatant is centrifuged at 50,000 g for 10 min. The supernatantis decanted, the pellet is resuspended by Polytron homogenization, asample is taken for BCA protein assay (Pierce, Rockford, Ill.), and thetissue is again centrifuged at 50,000 g. Pellets can be stored frozen at−20° C.

Radioligand Binding

For saturation binding, increasing concentrations of an appropriateligand such as [³H]Nα methylhistamine (70–90 Ci/mmol; Amersham PharmaciaBiotech, Piscataway, N.J.) are incubated without and with an appropriateconcentration of an appropriate agent such as 10⁻⁵ M thioperamide intriplicate with 40 to 60 μg of membrane protein in a total volume of 200μl of 50 mM Na-phosphate, pH 7.5, for 40 min at 24° C. The boundradioactivity is separated by filtration through Unifilter-96 GF/Bfilters (Packard, Meriden, Conn.) pretreated with 0.1% polyethyleneimine(Sigma). The filters are washed eight times with 400 μl of ice-cold 50mM Tris-HCl (pH 7.5), and radioactivity retained on the filters isquantitated by liquid scintillation counting in a Top-count (Packard) at34% efficiency. For competition binding assays, five concentrations ofcompounds are incubated in triplicate with an appropriate concentrationof an appropriate ligand such as 1 nM [³H]Nα methylhistamine and 70 μgof membrane protein under the conditions as described above. Samples arefiltered and radioactivity quantitated as described above. Binding datacan be analyzed by nonlinear least-squares curve-fitting to appropriatemodels with Prism software (GraphPad, San Diego, Calif.), and K_(i)values can be calculated from IC₅₀ values according to Cheng and Prusoff(1973).

Intracellular Calcium ([Ca²⁺]_(i)) Mobilization Assay

Cells are harvested 24 h post-transfection without trypsin and seeded at2.5×10⁵ cells/well in DMEM with 10% fetal bovine serum inpoly(D-lysine)-treated 96 well clear bottom black plates (BectonDickinson, Franklin Lakes, N.J.). Experimental compounds are diluted inHanks' balanced salt solution, 20 mM HEPES, 2.5 mM probenecid, 1% bovineserum albumin (wash buffer). Forty-eight hours post-transfection, cellsare loaded for 1.5 h with 2 μM Fluo 3-AM (F-6142; Sigma), 2.5 mMprobenecid, and 20 mM HEPES in DMEM with 10% fetal calf serum. Cells arewashed extensively with wash buffer to remove excess dye and evaluatedfor ligand-induced [Ca²⁺]_(i) release using the fluorometric imagingplate reader (FLIPR) (Molecular Devices, Sunnyvale, Calif.). Results aregiven as the relative change in fluorescence from the initial readingand measured over 3-min period following addition of compound.

cAMP Assay

Cells are transfected as previously described and assayed 48 hpost-transfection. Cells that are subjected to pertussis toxinpretreatment are incubated overnight before the assay with pertussistoxin (100 ng/ml) in full serum media. On the day of the assay cells areharvested in 2 mM EDTA/PBS and resuspended to a final concentration of5×10⁶ cells/ml in cold (4° C.) adenylate cyclase buffer (AC buffer) (250mM sucrose, 75 mM Tris-HCl, 12.5 mM MgCl₂, 1.5 mM EDTA, pH 7.4) to whichascorbic acid (10 mg/50 ml) and dithiothreitol (31 mg/50 ml) are addedfresh daily. The phosphodiesterase inhibitor Ro 20-1724(4-[(3-butoxy-4-methoxyphenyl) methyl]-2-imidazolidinone) is added at afinal concentration of 100 μM, and the cells are incubated for either 15min (room temperature) or 30 min (on ice). Drugs are prepared at 2×final concentrations in AC buffer±forskolin (10 μM for Chinese hamsterovary cells; 100 nM for the HEK-293 cells). For the assay, 50 μl of drugsolution is added to 50 μl of cell suspension in a 1 ml×96-well assayblock, incubated at 37° C. in an incubator-shaker for 15 min, boiled for3 min, and then cooled on ice. The cell lysates are then assayed fortotal cyclic AMP using the NEN cyclic AMP Flashplate Assay (New EnglandNuclear Life Science Products, Inc., Boston, Mass.) according to themanufacturer's protocol. Total cAMP produced for each condition isdetermined as follows: % B/Bo for each sample=(average net counts forsample of standard×100)/average net counts of zero standard. A standardcurve can be generated by plotting the % B/Bo for each standard versuslog[pmol of cAMP]. The concentration of cAMP for each sample can beinterpolated from the standard curve. Results are expressed asfemtomoles of cAMP/well.

MAP Kinase Assay

Cells are transfected as described above. Twenty-four hourspost-transfection, cells are harvested and re-seeded at a density of1×10⁶ cells/well in six-well dishes. Full serum media is replaced 5 to 8h after seeding with 0.4% serum media overnight. Cells that aresubjected to pertussis toxin pretreatment are incubated overnight beforethe assay with pertussis toxin at 100 ng/ml in 0.5% serum media. Onehour before the drug challenge, cells are placed in media without serumto reduce background MAP kinase activation. Drug is then added at theappropriate concentration and incubated for 5 min at 37° C. Cells arethen washed once with cold PBS and lysed in 100 μl of cold lysis buffer[150 mM NaCl, 50 mM Tris pH 8.0, 5 mM EDTA pH 98.0, 10 mM NaF, 10 mMdibasic sodium pyrophosphate, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodiumdeoxycholate (RIPA)] containing one Complete protease inhibitor cocktailtable/50 ml (Roche Molecular Biochemicals, Indianapolis, Ind.). Celllysates are collected in microfuge tubes and spun at 13,000 g for 15 minat 4° C. to pellet cellular debris. The protein concentration of thelysates is determined using the BCA protein assay. Twenty micrograms ofprotein is added to an equal volume of 2×SDS polyacrylamide gelelectrophoresis sample buffer and boiled for 5 min, then separated on a10% Tris-glycine polyacrylamide gel (Novex, Carlsbad, Calif.). Proteinsin the gel are transferred to a nitrocellulose membrane in transferbuffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) using asemidry transfer apparatus (Bio-Rad, Hercules, Calif.). Membranes areincubated in blocking solution [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1%(v/v) Tween 20 (TTBS)] containing 5% (w/v) milk for 1 h or more at roomtemperature. Membranes are rinsed three times with TTBS then developedusing the PhosphoPlus p44/42 MAP Kinase (Thr202/Tyr204) Antibody Kit(Cell Signaling Technology, Inc., Beverly, Mass.) according to themanufacturer's instructions.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. An isolated polynucleotide encoding at least 8 contiguous amino acidsfrom SEQ ID NO: 24, wherein the sequence of at least 8 contiguous aminoacids comprises the proline residue at position 6 and the leucineresidue at position 7 of the amino acid sequence of SEQ ID NO:
 24. 2.The isolated polynucleotide of claim 1, wherein the sequence encoded bythe polynucleotide comprises at least 10 contiguous amino acids of thepolypeptide.
 3. The isolated polynucleotide of claim 2, wherein thesequence encoded by the polynucleotide comprises at least 15 contiguousamino acids of the polypeptide.
 4. An isolated polynucleotide encodingthe amino acid sequence of SEQ ID NO:
 24. 5. The polynucleotide of claim4, having the sequence of SEQ ID NO:
 23. 6. A recombinant expressionvector comprising the polynucleotide of claim
 4. 7. A host cellcomprising the recombinant expression vector of claim
 6. 8. An isolatedpolypeptide having at least 8 contiguous amino acids from SEQ ID NO: 24,wherein the sequence of at least 8 contiguous amino acids comprises theproline residue at position 6 and the leucine residue at position 7 ofthe amino acid sequence of SEQ ID NO:
 24. 9. The isolated polypeptide ofclaim 8 comprising the amino acid sequence of SEQ ID NO: 24.